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Structural insights into the redox-switch mechanism of the MarR/DUF24-type regulator HypR.

Palm GJ, Khanh Chi B, Waack P, Gronau K, Becher D, Albrecht D, Hinrichs W, Read RJ, Antelmann H - Nucleic Acids Res. (2012)

Bottom Line: HypR controls positively a flavin oxidoreductase HypO that confers protection against NaOCl stress.The crystal structures of reduced and oxidized HypR proteins were resolved revealing structural changes of HypR upon oxidation.In reduced HypR a hydrogen-bonding network stabilizes the reactive Cys14 thiolate that is 8-9 Å apart from Cys49'.

View Article: PubMed Central - PubMed

Affiliation: Institute for Biochemistry, Ernst-Moritz-Arndt-University of Greifswald, D-17487 Greifswald, Germany.

ABSTRACT
Bacillus subtilis encodes redox-sensing MarR-type regulators of the OhrR and DUF24-families that sense organic hydroperoxides, diamide, quinones or aldehydes via thiol-based redox-switches. In this article, we characterize the novel redox-sensing MarR/DUF24-family regulator HypR (YybR) that is activated by disulphide stress caused by diamide and NaOCl in B. subtilis. HypR controls positively a flavin oxidoreductase HypO that confers protection against NaOCl stress. The conserved N-terminal Cys14 residue of HypR has a lower pK(a) of 6.36 and is essential for activation of hypO transcription by disulphide stress. HypR resembles a 2-Cys-type regulator that is activated by Cys14-Cys49' intersubunit disulphide formation. The crystal structures of reduced and oxidized HypR proteins were resolved revealing structural changes of HypR upon oxidation. In reduced HypR a hydrogen-bonding network stabilizes the reactive Cys14 thiolate that is 8-9 Å apart from Cys49'. HypR oxidation breaks these H-bonds, reorients the monomers and moves the major groove recognition α4 and α4' helices ∼4 Å towards each other. This is the first crystal structure of a redox-sensing MarR/DUF24 family protein in bacteria that is activated by NaOCl stress. Since hypochloric acid is released by activated macrophages, related HypR-like regulators could function to protect pathogens against the host immune defense.

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Structures of reduced HypRC14S (A) and oxidized HypR proteins (B), superimposition of reduced and oxidized HypR (C) and secondary structure assignments (D). (A) Reduced HypRC14S dimer with monomers C and D coloured in grey and blue, respectively. Residues Ser14 and Cys49 are shown in yellow as sticks and spheres (Oγ and Sγ, respectively). (B) Oxidized HypR protein with the Cys14–Cys49′ intersubunit disulphide with monomers C and D, labelled in grey and blue, respectively. The intermolecular disulphide bond is shown in yellow in monomer C. (C) Superimposition of the oxidized HypR dimer (light and dark blue) and the reduced HypR dimer (light and dark green). The HypRox/red side view is shown and Cys14 and Cys49′ are shown as yellow sticks. One monomer of each dimer (on the right, dark blue and dark green) is aligned to visualize the differences in the opposing monomers. (D) The secondary structure elements of HypR are α1(15–23), α2(28–35), α3(42–48), α4(54–66), β2(70–75), β3(81–86), α5(88–114) that are shown as red tubes (α-helices) and green arrows (β-sheets). The redox-sensing Cys14 and the Cys49 are labelled in yellow. (E-H) The pocket of the redox-sensitive Cys residues in reduced and oxidized HypR. (E) Electron density map (2Fo–Fc at 1σ in blue) and (F) key interactions between α1 and α3′ helices including the Cys14Ser and Cys49′ residues for reduced HypR. The atoms are coloured in yellow (carbon), dark blue (nitrogen), red (oxygen) and green (sulphur); water molecules are shown as red spheres and hydrogen bonds are labelled as dashed lines. Cys14Ser is shown in orange forming hydrogen bonds with Val16 and Glu17. Cys49′ is in a hydrophobic environment formed by Ile52′, Trp27′, Ile30′, Leu31′, Gln60Cγ′, Ile48′. The distance between Ser14Oγ and Cys49′Sγ that form the intermolecular disulphide bond is 8.48 Å. (G) Electron density map (2Fo–Fc at 1σ in blue) and (H) structure of the Cys14–Cys49′ intersubunit disulphide region in oxidized HypR. Helices α3′ and α4′ on the right are oriented as in Figure 7EF, this emphasizes the movement of α4 on the left.
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gkr1316-F7: Structures of reduced HypRC14S (A) and oxidized HypR proteins (B), superimposition of reduced and oxidized HypR (C) and secondary structure assignments (D). (A) Reduced HypRC14S dimer with monomers C and D coloured in grey and blue, respectively. Residues Ser14 and Cys49 are shown in yellow as sticks and spheres (Oγ and Sγ, respectively). (B) Oxidized HypR protein with the Cys14–Cys49′ intersubunit disulphide with monomers C and D, labelled in grey and blue, respectively. The intermolecular disulphide bond is shown in yellow in monomer C. (C) Superimposition of the oxidized HypR dimer (light and dark blue) and the reduced HypR dimer (light and dark green). The HypRox/red side view is shown and Cys14 and Cys49′ are shown as yellow sticks. One monomer of each dimer (on the right, dark blue and dark green) is aligned to visualize the differences in the opposing monomers. (D) The secondary structure elements of HypR are α1(15–23), α2(28–35), α3(42–48), α4(54–66), β2(70–75), β3(81–86), α5(88–114) that are shown as red tubes (α-helices) and green arrows (β-sheets). The redox-sensing Cys14 and the Cys49 are labelled in yellow. (E-H) The pocket of the redox-sensitive Cys residues in reduced and oxidized HypR. (E) Electron density map (2Fo–Fc at 1σ in blue) and (F) key interactions between α1 and α3′ helices including the Cys14Ser and Cys49′ residues for reduced HypR. The atoms are coloured in yellow (carbon), dark blue (nitrogen), red (oxygen) and green (sulphur); water molecules are shown as red spheres and hydrogen bonds are labelled as dashed lines. Cys14Ser is shown in orange forming hydrogen bonds with Val16 and Glu17. Cys49′ is in a hydrophobic environment formed by Ile52′, Trp27′, Ile30′, Leu31′, Gln60Cγ′, Ile48′. The distance between Ser14Oγ and Cys49′Sγ that form the intermolecular disulphide bond is 8.48 Å. (G) Electron density map (2Fo–Fc at 1σ in blue) and (H) structure of the Cys14–Cys49′ intersubunit disulphide region in oxidized HypR. Helices α3′ and α4′ on the right are oriented as in Figure 7EF, this emphasizes the movement of α4 on the left.

Mentions: To avoid artificial oxidation of HypR during crystallization, HypRC14S protein was crystallized in the presence of β-mercaptoethanol. The structure was solved using molecular replacement and refined to 1.8 Å resolution. Electron density is visible for residues 13–117 of the native 125 residues (Figure 7). The asymmetric unit contains four similar monomers, organized in 2 biological dimers, 4 mercaptoethanol and 279 water molecules. The structure was refined to an Rcryst of 0.1935 and Rfree of 0.2336. The overall structure of reduced HypR comprises a homodimer adopting a triangular shape with non-crystallographic 2-fold symmetry as found in all MarR-family proteins (48). The structure has the following secondary structure elements: α1(15–23)–α2(28–35)–α3(42–48)–α4(54–66)–β2(70–75)–β3(81–86)–α5(88–114) (Figure 7A and D). The two monomers associate via a large dimer interface of 1600 Å2 provided by helices α1, α2, α5 and their symmetry mates α1′, α2′, α5′. Helix α5 is much longer than in the OhrR-family proteins exemplified by SarZ of S. aureus and OhrR of X. campestris (Supplementary Figure S1B). Pro95 and Gly105 induce kinks at positions 93 and 107 in HypR. The dimer interface significantly differs from that in OhrR structures since α6 is missing in HypR (Supplementary Figures S1B, S7A, S7C and S7D).Figure 7.


Structural insights into the redox-switch mechanism of the MarR/DUF24-type regulator HypR.

Palm GJ, Khanh Chi B, Waack P, Gronau K, Becher D, Albrecht D, Hinrichs W, Read RJ, Antelmann H - Nucleic Acids Res. (2012)

Structures of reduced HypRC14S (A) and oxidized HypR proteins (B), superimposition of reduced and oxidized HypR (C) and secondary structure assignments (D). (A) Reduced HypRC14S dimer with monomers C and D coloured in grey and blue, respectively. Residues Ser14 and Cys49 are shown in yellow as sticks and spheres (Oγ and Sγ, respectively). (B) Oxidized HypR protein with the Cys14–Cys49′ intersubunit disulphide with monomers C and D, labelled in grey and blue, respectively. The intermolecular disulphide bond is shown in yellow in monomer C. (C) Superimposition of the oxidized HypR dimer (light and dark blue) and the reduced HypR dimer (light and dark green). The HypRox/red side view is shown and Cys14 and Cys49′ are shown as yellow sticks. One monomer of each dimer (on the right, dark blue and dark green) is aligned to visualize the differences in the opposing monomers. (D) The secondary structure elements of HypR are α1(15–23), α2(28–35), α3(42–48), α4(54–66), β2(70–75), β3(81–86), α5(88–114) that are shown as red tubes (α-helices) and green arrows (β-sheets). The redox-sensing Cys14 and the Cys49 are labelled in yellow. (E-H) The pocket of the redox-sensitive Cys residues in reduced and oxidized HypR. (E) Electron density map (2Fo–Fc at 1σ in blue) and (F) key interactions between α1 and α3′ helices including the Cys14Ser and Cys49′ residues for reduced HypR. The atoms are coloured in yellow (carbon), dark blue (nitrogen), red (oxygen) and green (sulphur); water molecules are shown as red spheres and hydrogen bonds are labelled as dashed lines. Cys14Ser is shown in orange forming hydrogen bonds with Val16 and Glu17. Cys49′ is in a hydrophobic environment formed by Ile52′, Trp27′, Ile30′, Leu31′, Gln60Cγ′, Ile48′. The distance between Ser14Oγ and Cys49′Sγ that form the intermolecular disulphide bond is 8.48 Å. (G) Electron density map (2Fo–Fc at 1σ in blue) and (H) structure of the Cys14–Cys49′ intersubunit disulphide region in oxidized HypR. Helices α3′ and α4′ on the right are oriented as in Figure 7EF, this emphasizes the movement of α4 on the left.
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gkr1316-F7: Structures of reduced HypRC14S (A) and oxidized HypR proteins (B), superimposition of reduced and oxidized HypR (C) and secondary structure assignments (D). (A) Reduced HypRC14S dimer with monomers C and D coloured in grey and blue, respectively. Residues Ser14 and Cys49 are shown in yellow as sticks and spheres (Oγ and Sγ, respectively). (B) Oxidized HypR protein with the Cys14–Cys49′ intersubunit disulphide with monomers C and D, labelled in grey and blue, respectively. The intermolecular disulphide bond is shown in yellow in monomer C. (C) Superimposition of the oxidized HypR dimer (light and dark blue) and the reduced HypR dimer (light and dark green). The HypRox/red side view is shown and Cys14 and Cys49′ are shown as yellow sticks. One monomer of each dimer (on the right, dark blue and dark green) is aligned to visualize the differences in the opposing monomers. (D) The secondary structure elements of HypR are α1(15–23), α2(28–35), α3(42–48), α4(54–66), β2(70–75), β3(81–86), α5(88–114) that are shown as red tubes (α-helices) and green arrows (β-sheets). The redox-sensing Cys14 and the Cys49 are labelled in yellow. (E-H) The pocket of the redox-sensitive Cys residues in reduced and oxidized HypR. (E) Electron density map (2Fo–Fc at 1σ in blue) and (F) key interactions between α1 and α3′ helices including the Cys14Ser and Cys49′ residues for reduced HypR. The atoms are coloured in yellow (carbon), dark blue (nitrogen), red (oxygen) and green (sulphur); water molecules are shown as red spheres and hydrogen bonds are labelled as dashed lines. Cys14Ser is shown in orange forming hydrogen bonds with Val16 and Glu17. Cys49′ is in a hydrophobic environment formed by Ile52′, Trp27′, Ile30′, Leu31′, Gln60Cγ′, Ile48′. The distance between Ser14Oγ and Cys49′Sγ that form the intermolecular disulphide bond is 8.48 Å. (G) Electron density map (2Fo–Fc at 1σ in blue) and (H) structure of the Cys14–Cys49′ intersubunit disulphide region in oxidized HypR. Helices α3′ and α4′ on the right are oriented as in Figure 7EF, this emphasizes the movement of α4 on the left.
Mentions: To avoid artificial oxidation of HypR during crystallization, HypRC14S protein was crystallized in the presence of β-mercaptoethanol. The structure was solved using molecular replacement and refined to 1.8 Å resolution. Electron density is visible for residues 13–117 of the native 125 residues (Figure 7). The asymmetric unit contains four similar monomers, organized in 2 biological dimers, 4 mercaptoethanol and 279 water molecules. The structure was refined to an Rcryst of 0.1935 and Rfree of 0.2336. The overall structure of reduced HypR comprises a homodimer adopting a triangular shape with non-crystallographic 2-fold symmetry as found in all MarR-family proteins (48). The structure has the following secondary structure elements: α1(15–23)–α2(28–35)–α3(42–48)–α4(54–66)–β2(70–75)–β3(81–86)–α5(88–114) (Figure 7A and D). The two monomers associate via a large dimer interface of 1600 Å2 provided by helices α1, α2, α5 and their symmetry mates α1′, α2′, α5′. Helix α5 is much longer than in the OhrR-family proteins exemplified by SarZ of S. aureus and OhrR of X. campestris (Supplementary Figure S1B). Pro95 and Gly105 induce kinks at positions 93 and 107 in HypR. The dimer interface significantly differs from that in OhrR structures since α6 is missing in HypR (Supplementary Figures S1B, S7A, S7C and S7D).Figure 7.

Bottom Line: HypR controls positively a flavin oxidoreductase HypO that confers protection against NaOCl stress.The crystal structures of reduced and oxidized HypR proteins were resolved revealing structural changes of HypR upon oxidation.In reduced HypR a hydrogen-bonding network stabilizes the reactive Cys14 thiolate that is 8-9 Å apart from Cys49'.

View Article: PubMed Central - PubMed

Affiliation: Institute for Biochemistry, Ernst-Moritz-Arndt-University of Greifswald, D-17487 Greifswald, Germany.

ABSTRACT
Bacillus subtilis encodes redox-sensing MarR-type regulators of the OhrR and DUF24-families that sense organic hydroperoxides, diamide, quinones or aldehydes via thiol-based redox-switches. In this article, we characterize the novel redox-sensing MarR/DUF24-family regulator HypR (YybR) that is activated by disulphide stress caused by diamide and NaOCl in B. subtilis. HypR controls positively a flavin oxidoreductase HypO that confers protection against NaOCl stress. The conserved N-terminal Cys14 residue of HypR has a lower pK(a) of 6.36 and is essential for activation of hypO transcription by disulphide stress. HypR resembles a 2-Cys-type regulator that is activated by Cys14-Cys49' intersubunit disulphide formation. The crystal structures of reduced and oxidized HypR proteins were resolved revealing structural changes of HypR upon oxidation. In reduced HypR a hydrogen-bonding network stabilizes the reactive Cys14 thiolate that is 8-9 Å apart from Cys49'. HypR oxidation breaks these H-bonds, reorients the monomers and moves the major groove recognition α4 and α4' helices ∼4 Å towards each other. This is the first crystal structure of a redox-sensing MarR/DUF24 family protein in bacteria that is activated by NaOCl stress. Since hypochloric acid is released by activated macrophages, related HypR-like regulators could function to protect pathogens against the host immune defense.

Show MeSH